The present invention relates to enzymes having the function of decomposing, using microorganisms, thiophene compounds, namely benzothiophene, dibenzothiophene (hereinafter referred to as xe2x80x9cDBTxe2x80x9d) and their substituted compounds, or derivatives thereof, and genes encoding the enzymes. By using the enzymes and the gene defined in the present invention, sulfur can be released from benzothiophene, DBT and their substituted compounds, or derivatives thereof which are contained in fossil fuels such as petroleum. As a result, sulfur, which is generally diffused in the air when fossil fuels such as petroleum and coal are burned, can be easily removed from the fossil fuel.
In order to remove sulfur from hydrocarbon fuel such as petroleum, methods including alkali treating or solvent desulfurization are known. However, at present, mainly hydrodesulfurization is used. Hydrodesulfurization is a method for reacting sulfur compounds in a petroleum fraction with hydrogen in the presence of a catalyst and removing the produced hydrogen sulfide so as to obtain low-sulfur products. As a catalyst, metallic catalysts such as cobalt, molybdenum, nickel and tungsten are used with alumina as a carrier. When the molybdenum on alumina is used as the catalyst, usually cobalt or nickel is added as a promoter to enhance catalysis performance. The hydrodesulfurization with metallic catalysts is undoubtedly a fine process which is widely used throughout the world at the moment. However, as a process for producing petroleum products adapted to more strict environmental regulations, there are some problems. Some examples are discussed below briefly.
Generally the substrate specificity of a metallic catalyst is low, and so it is suitable for decomposing various kinds of sulfur compounds and lowering the amount of sulfur contained in the fossil fuel as a whole. However, it is considered that the effect of desulfurization with metallic catalyst is sometimes insufficient for a specific group of sulfur compounds, i.e., heterocyclic sulfur compounds such as benzothiophene, DBT and their alkyl derivatives. For example, after desulfurizing light oil, various heterocyclic organic sulfur compounds still remain. One reason why the effect of desulfurization with metallic catalyst is insufficient would be steric hindrance caused by substituents which are around the sulfur atoms of the organic sulfur compounds. Among these substituted compounds, the influence of a methyl substituted compound on the reaction of a metallic catalyst has been studied in relation to thiophene, benzothiophene, DBT and so on. According to such studies, it is generally said that, as the number of substituted compounds increases, desulfurization reaction rates decreases. However, it is also said that the position of the substituents have a very large influence on the reactivity. One of the reports which have shown that the steric hindrance has the significant influence on the reaction of metallic catalyst is, for example, Houalla, M., Broderick, D. H., Sapre, A. V., Nag, N. K., de Beer, V. H., Gates, B. C., Kwart, H. J., Catalt., 61, 523-527(1980). In fact, it is known that a considerable amount of various alkyl derivatives of DBT exists in light oil (e.g. Kabe, T., Ishihara, A. and Tajima, H. Ind. Eng. Chem. Res., 31, 1577-1580(1992)).
As stated above, it is considered that, in order to desulfurize organic sulfur compounds which are resistant against hydrodesulfurization, higher reaction temperature and pressure than that usually used are required, and also the amount of hydrogen added to be increased remarkably. It is thus expected that enormous capital investment and operating costs are needed to improve a hydrodesulfurization process such as this. For example, light oil contains organic sulfur compounds resisting such hydrodesulfurization as a major compound species, and as stated above, a substantial improvement on the hydrodesulfurization process is required to carry out more sophisticated desulfurization of light oil (an ultra deep desulfurization).
On the other hand, the enzyme-reaction in an organism proceeds under relatively mild conditions, and further, the rate of enzyme reaction in an organism compares favorably with that of a chemical catalyst. Moreover, there are so many kinds of enzymes in vivo to conform appropriately to various kinds of vital reactions occurring therein, and those enzymes usually show a very high substrate specificity. These characteristics are expected to be utilized for so-called biodesulfurization reaction, which removes sulfur from sulfur compounds in fossil fuel by using microorganisms (Monticello, D. J., Hydrocarbon Processing 39-45(1994)).
There are a large number of reports on methods for removing sulfur from heterocyclic sulfur compounds which are ingredients of petroleum by using bacteria, and these methods are broadly divided into the reaction of decomposing a ring (Cxe2x80x94C bond cleavage) and the Cxe2x80x94S bond cleavage reaction. As bacteria having Cxe2x80x94C-bondxe2x80x94attacking desulfurization activity, for example, strains belonging to Pseudomonas sp., Pseudomonas aeruginosa, Beijerinckia sp., Pseudomonas alcaligenes, Pseudomonas stutzeri, Pseudomonas putida, Brevibacterium sp. are known. These bacteria carry out the cleavage of Cxe2x80x94C bond in heterocyclic sulfur compounds of which a representative example is DBT, decompose a benzene ring, thereafter, by oxidative reaction cascade, they conduct a metabolism in which salt containing sulfur atom(s) is released. As the reaction mechanism of the carbon-backbone-attacking pathway, there are the hydroxylation of aromatic ring (DBTxe2x86x92xe2x86x921,2-dihydroxy DBT), the cleavage of a ring, and the oxidation to water-soluble product (1,2-dihydroxy DBTxe2x86x92xe2x86x92trans-4 [2-(3-hydroxy) thianaphthenyl]-2-oxo-butenoic acid, 3-hydroxy-2-formylbenzothiophene), and this reaction mechanism is called xe2x80x9cKodama pathwayxe2x80x9d. The Cxe2x80x94C bond in a benzene ring of DBT is attacked by this kind of reaction to generate various water-soluble substances which are extractable from the oil. Due to this reaction, however, other aromatic molecules in the oil are also attacked, and as a result, a significant amount of hydrocarbons move to water phase (Hartdegen, F. J., Coburn, J. M. and Roberts, R. L. Chem. Eng. Progress, 80, 63-67(1984)). This causes the reduction of total calories of petroleum and so it is an industrially ineffective reaction. Furthermore, as Kodama et al. has reported, this type of bacteria oxidatively decomposing DBT provides water-soluble thiophene compounds (mainly 3-hydroxy-2-formylbensothiophene) as oxidized products, but this is a substance difficult to remove from water phase. In addition, since the attack to the carbon ring of DBT often occurs at position 2 or 3 of DBT, DBT substituted with an alkyl or alkyl groups at these positions does not become the substrate of the Kodama pathway.
It has been reported that there are microorganisms which decompose not only crude oil or coal but also model compounds containing sulfur, remove selectively hetero-atom sulfur, and generate sulfate and hydroxyl compounds. Taking the structure of the metabolites into consideration, this kind of reaction is considered to be one which cleaves specifically Cxe2x80x94S bond in sulfur compounds and accordingly releases sulfur in the form of sulfate. As shown in Table 1, to date, some biodesulfurization reaction systems which are characterized by attacking sulfur have been reported.
For all biodesulfurizations stated above, a metabolic reaction of microorganism cultured at around 30xc2x0 C. is used. On the other hand, it is known that generally the rate of chemical reaction increases as the temperature becomes higher. Regarding the desulfurization in petroleum refining process, fractional distillation or desulfurization reaction is carried out under conditions of high temperature and high pressure. Therefore, when biodesulfurization is incorporated into the petroleum refining process, it is desirable that the desulfurization reaction is carried out at higher temperature in the mid course of cooling process, without cooling the fraction to room temperature. Some reports on high-temperature biodesulfurization are as follows.
Most attempts to carry out the desulfurization reaction using microorganisms at room temperature are directed to coal desulfurization. Coal contains various kinds of sulfur compounds. The main inorganic sulfur compound is pyrite. On the other hand, the organic sulfur compounds vary widely in type, and it is known that the majority of these contain thiol, sulfide, disulfide and thiophene groups. The microorganisms used are Sulfolobus bacteria which are all thermophiles. There are several reports that various Sulfolobus strains were used in the leaching of metal out of mineral sulfide (Brierley C. L. and Murr, L. E., Science 179, 448-490(1973)), the desulfurization of pyrite in coal (Kargi, F. and Robinson, J. M., Biotechnol. Bioeng, 24, 2115-2121(1982); Kargi, F. and Robinson, J. M., Appl. Environ. Microbiol., 44, 878-883(1982); Kargi, F. and Cervoni, T. D., Biotechnol. Letters 5, 33-38(1983); Kargi, F. and Robinson, J. M., Biotechnol. Bioeng., 26, 687-690(1984); Kargi, F. and Robinson, J. M., Biotechnol. Bioeng. 27, 41-49(1985); Kargi, F., Biotechnol. Lett., 9, 478-482(1987)) and so on. According to Kargi and Robinson (Kargi, F and Robinson, J. M., Appl. Environ. Microbiol., 44, 878-883(1982)), a certain strain of Sulfolobus acidocaldarius isolated from an acidic thermal spring of Yellowstone National Park in U.S.A. grows at 45-70xc2x0 C. and oxidizes elemental sulfur at an optimum pH2. Furthermore, it has been also reported that two other kinds of Sulfolobus acidocaldarius stains oxidize pyrite (Tobita, M., Yokozeki, M., Nishikawa, N. and Kawakami, Y., Biosci. Biotech. Biochem. 58, 771-772(1994)).
It is known that, among the organic sulfur compounds contained in fossil fuel, DBT and its substituted compounds, or derivatives thereof, are generally resistant to hydrodesulfurization in the petroleum refining process. High-temperature decomposition by Sulfolobus acidocaldaius (hereinafter, referred to as xe2x80x9cS. acidocaldariusxe2x80x9d) of the said DBT has been also reported (Kargi, and Robinson, J. M., Biotechnol. Bioeng, 26, 687-690(1984); Kargi, F., Biotechnol. Letters 9, 478-482(1987)).
According to these reports, when model aromatic heterocyclic sulfur compounds such as thianthrene, thioxanthene, DBT and the like are reacted with S. acidocaldarius at high temperature, these sulfur compounds are oxidized and decomposed. Oxidation of these aromatic heterocyclic sulfur compounds by this microorganism is observed at 70xc2x0 C. and it results in the formation of sulfate ions as the reaction product. However, because this reaction is carried out in a medium which does not contain any carbon source other than sulfur compounds, these sulfur compounds would be also used as the carbon sources. That is to say, it is clear that Cxe2x80x94C bond in sulfur compounds was decomposed. Furthermore, S. acidocaldarius can be grown only in an acidic medium, and the oxidative decomposition reaction require under severely acidic conditions (e.g. pH2.5) to continue. Since such conditions cause the degradation of petroleum products and at the same time requires acid-resistant materials in the desulfurization-associated step, it is considered not to be desirable for the process. When S. acidocaldarius is grown under autotrophic conditions, the microorganism acquires necessary energy from reduced iron-sulfur compounds and uses carbon dioxide as the carbon source. Alternatively, when S. acidocaldarius is grown under heterotrophic conditions, it can use various organic compounds as carbon and energy sources. In other words, it can be said when fossil fuel exists, it can be used as a carbon source.
Finnerty et al. has reported that the strains belonging to Pseudomonas stutzeri, Pseudomonas alcaligenes and Pseudomonas putida decompose DBT, benzothiophene, thioxanthene and thianthrene, and convert them into water-soluble substances (Finnerty, W. R., Shockiey, K., Attaway, H. in Microbial Enhanced Oil Recovery, Zajic, J. E. et al.(eds.) Penwell Tuisa, Okia, 83-91(1983)). In this case, the oxidative reaction can proceed at 55xc2x0 C. However, the decomposed products of DBT by these Pseudomonas strains are 3-hydroxy-2-formylbenzothiophene reported by Kodama et al. (Monticello, D. J., Bakker, D., Finnerty, W. R. Appl. Environ. Microbiol., 49, 756-760(1985)). The oxidation activity of DBT by the Pseudomonas strains is induced by an aromatic hydrocarbon without sulfur such as naphthalene or salicylic acid, and is blocked by chloramphenicol. From this fact, it was found that the decomposition reaction of DBT by the Pseudomonas strains is based on the cleavage of a Cxe2x80x94C bond in aromatic ring. Moreover, there is the risk that valuable aromatic hydrocarbons other than sulfur compounds in the petroleum fraction are also decomposed together with them, and if this occurs, it results in lowering of fuel value or petroleum fraction quality.
As stated above, the known strains which can decompose DBT at high temperature are the ones which catalyze the reaction of cleaving a Cxe2x80x94C bond in the DBT molecule and use the resulting compounds as carbon sources. As mentioned above, the decomposition reaction of organic sulfur compounds which cleaves specifically Cxe2x80x94S bond but leaves Cxe2x80x94C bond unchangeable is desirable as a real method for desulfurizing petroleum. In other words, the most desirable biodesulfurization process is one which has an activity of cleaving Cxe2x80x94S bond in the molecule of DBT and its alkyl-substituted compounds, or their derivatives at high temperature and uses microorganisms which generate desulfurization products in the form of water-soluble substances.
As stated above, several families of bacteria are known as microorganisms conducting the Cxe2x80x94S bond cleavage to decompose DBT. However, of all these bacteria, there were found no examples described to have an activity of decomposing DBT under high temperature conditions of more than 42xc2x0 C. For example, ATCC53968 (Rhodococcus sp). is a thoroughly studied DBT-decomposing strain and conducts an addition of an oxygen atom to the sulfur atom of DBT, generating DBT sulfone (hereinafter referred to as xe2x80x9cDBTO2xe2x80x9d) from DBT sulfoxide (hereinafter referred to as xe2x80x9cDBTOxe2x80x9d), and further generating 2-hydroxybiphenyl (hereinafter referred to as xe2x80x9c2-HBPxe2x80x9d) via 2-(2xe2x80x2-hydroxyphenyl) benzensulfinate. However, it has been reported that even this strain grows very slowly or stops growing, when it is cultured for 48 hours at a temperature of 37xc2x0 C. or 43xc2x0 C. which is slightly higher than 30xc2x0 C. (an ordinary culturing temperature) (Japanese Patent Application Laying-Open (kokai) No. 6-54695). Therefore, it has been presumed that the use of the microorganism, which can grow under high temperatures condition and can cleave specifically the Cxe2x80x94S bond of heterocyclic sulfur compounds including organic sulfur compounds, especially DBT, its substituted compounds, or their derivatives at high temperature, is more suitable for conducting the desulfurization reaction at high temperature. The present inventors have conducted a wide range of screenings, has amplified the microorganisms under high temperature conditions, nearly 60xc2x0 C., and has already isolated 2 strains of Paenibacillus sp., which are high-temperature desulfurizing strains having a function of decomposing and desulfurizing DBT families for the first time in the world (Japanese Patent Application Laying-Open (kokai) No. 10-036859). If genes which are associated with high-temperature desulfurization activity can be isolated from this strain, it is possible to endow a wide range of microbes with the function of high-temperature desulfurization by using genetic engineering such as recombinant DNA technology.
Among the bacteria known for their function of conducting Cxe2x80x94S bond cleavages in the decomposition reaction, genes thereof, which encode enzyme activities involved in DBT decomposition reaction that are identified and whose nucleotide sequences are determined are, to the best of the present inventors"" knowledge, only dsz genes of Rhodococcus sp. IGTS8 strain (Denome, S., Oldfleld., C., Nash, L. J. and Young, K. D. J.Bacteriol., 176:6707-6716, 1994; Piddington, C. S., Kovacevich, B. R. and Rambosek, J. Appl. Environ. Microbiol., 61:468-475, 1995). The DBT decomposition reaction by IGTS8 strain is catalyzed by three enzymes: DszC catalyzing the conversion from DBT to DBTO2 via DBTO, DszA catalyzing the conversion from DBTO2 to 2-(2xe2x80x2-hydroxyphenyl) benzensulfinic acid, and DszB catalyzing the conversion from 2-(2xe2x80x2-hydroxyphenyl) benzensulfinic acid to 2-HBP (Denome, S., Oldfield., C., Nash, L. J. and Young, K. D. J.Bacteriol., 176:6707-6716, 1994; Gray, K. A., Pogrebinshy, O. S., Mrachko, G. T., Xi, L. Monticello, D. J. and Squires, C. H. Nat Biotechnol., 14:1705-1709, 1996; Oldfield, C., Pogrebinsky, O., Simmonds, J., Olson, E. S. and Kulpa, C. F., Microbiology, 143:2961-2973, 1997). The genes corresponding to the above enzymes are called dszA, dszB and dszC. It is known that the enzymes DszC and DszA are monooxygenases, and both enzymes need the coexistence of NADH-FMN oxidoreductase activity for their oxygenation reaction (Gray, K. A., Pogrebinsky, O. S., Mrachko, G. T., Xi, L. Monticello, D. J. and Squires, C. H. Nat Biotechnol., 14:1705-1709, 1996; Xi, L. Squires, C. H., Monticello, D. J. and Childs, J. D. Biochem. Biophys. Res Commun., 230:73-76, 1997) It has been reported that when the dsz genes are induced and expressed in Escherichia coli by shifting the temperature, DszA activity by cell culture reaches the maximum at 39xc2x0 C., but remarkably decreases at 42xc2x0 C. (Denome, S., Oldfield., D., Nash, L. J. and Young, K. D. J. Bacteriol., 176:6707-6716, 1994). This report corresponds to the result of an experiment on resting cell reaction system which shows that the desulfurization enzyme activity of IGTS8 strain reaches the maximum around room temperature, but activity decreases at higher temperature and there is no desulfurization activity at temperatures of more than 50xc2x0 C. (Konishi, J., Ishii, Y., Onaka, T., Okumura, K. and Suzuki, M. Appl. Environ. Microbiol., 63:3164-3169, 1997). Therefore, the genes which direct DBT-decomposing activity specific for Cxe2x80x94S bond under high temperature conditions, more than 50xc2x0 C., have not been previously reported.
One object of the present invention is to isolate the genes involved in high-temperature desulfurization reaction from a microorganism having an ability of acting on benzothiophene and DBT compounds and decomposing them at high temperature, to specify the structure (especially the nucleotide sequences), and to create novel desulfurizing microorganisms by introducing the genes into a heterologous microorganism and endowing it with the ability of desulfurization. Another object of the present invention is to establish a method for removing sulfur by actually contacting such a microorganism with benzothiophene, DBT and their alkyl derivatives and cleaving the Cxe2x80x94S bonds of these compounds.
After thorough studies directed to achieve the above objects, the present inventors have succeeded in isolating the genes involved in desulfurization reaction from high-temperature desulfurization bacteria, Paenibacillus sp., and have completed the present invention.
That is to say, the first aspect of the present invention relates to genes encoding desulfurization enzymes.
The second aspect of the present invention relates to vectors containing the said genes.
The third aspect of the present invention relates to transformants containing the said vectors.
The forth aspect of the present invention relates to desulfurization enzymes.
The fifth aspect of the present invention relates to genes encoding transposase.
The sixth aspect of the present invention relates to transposase.
This specification includes part or all of the contents as disclosed in the specifications and/or drawings of Japanese Patent Application Nos. 10-090387 and 10-310545 which are priority documents of the present application.
The details of the present invention are disclosed below.
(1) Gene Encoding a Desulfurization Enzyme
The genes of the present invention comprise the following three types of genes.
The first gene encodes (a) a protein represented by an amino acid sequence shown in SEQ ID NO: 2; or (b) a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2, and having a function of converting DBTO2 into 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid.
The second gene encodes (a) a protein represented by an amino acid sequence shown in SEQ ID NO: 4; or (b) a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 4, and having a function of converting 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid into 2-HBP.
The third gene encodes (a) a protein represented by an amino acid sequence shown in SEQ ID NO: 6; or (b) a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 6, and having a function of converting DBT into DBTO2 via DBTO.
The above-described first, second and third genes have a certain homology to dszA, dszB or dszC derived from Rhodococcus sp. IGTS8 strain. However, the proteins encoded by these genes are different from the ones encoded by dszA, dszB and dszC in terms of their properties.
Among the genes of the present invention, the ones which encode amino acid sequences as shown in SEQ ID NOS: 2, 4 and 6 can be obtained by the methods described later in Examples. Since the nucleotide sequences of these genes have been already determined as shown in SEQ ID NOS: 1, 3 and 5, they can also be obtained by synthesizing primers on the basis of these nucleotide sequences, and carrying out PCR using the primers and a DNA as a template, the DNA being prepared from Paenibacillus sp. A11-1 strain (which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology under accession No. FERM BP-6025 on Jul. 22, 1997) or A11-2 strain (which was deposited with the same international depositary authority under accession No. FERM BP-6026 on Jul. 22, 1997).
The genes encoding amino acid sequences comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NOS: 2, 4 and 6 can be obtained by modifying the genes encoding amino acid sequences shown in SEQ ID NOS: 2, 4 and 6, by techniques in common use at the time of the filing date of the present application, for example site-directed mutagenesis (Zoller et al., Nucleic Acids Res. 10: 6487-6500, 1982.
Since the genes of the present invention encode enzymes which are associated with the decomposition of DBT, they can be used to desulfurize petroleum.
(2) Vector Comprising a Gene Which Encodes a Desulfurization Enzyme
The vector of the present invention comprises the above-described first, second or third gene. Such a vector can be prepared by inserting a DNA fragment containing the first, second or third gene of the present invention into a known vector. The vector into which the DNA fragment is inserted is determined depending on the type of host being transformed. If Escherichia coli is used as the host, the following vector can preferably be used. It is preferable to use vectors such as pUR, pGEX, pUC, pET, pT7, pBluescript, pKK, pBS, pBC, pCAL and the like, which carry lac, lacUV5, trp, tac, trc, xcexpL, T7, rrnB or the like as a strong promoter.
(3) Transformant Comprising a Vector Containing Genes Which Encode a Desulfurization Enzyme
The transformant of the present invention comprises a said vector. The cells used as a transformation host may be from a plant or animal, but microorganisms such as Escherichia coli are more preferable. Typical strains include, for example, 71/18, BB4, BHB2668, BHB2690, BL21(DE3), BNN102(C600hflA), C-1a, C600(BNN93), CES200, CES201, CJ236, CSH18, DH1, DH5, DH5 xcex1, DP50supF, ED8654, ED8767, HB101, HMS174, JM101, JM105, JM107, JM109, JM110, K802, KK2186, LE392, LG90, M05219, MBM7014.5, MC1061, MM294, MV1184, MV1193, MZ-1, NM531, NM538, NM539, Q358, Q359, R594, RB791, RR1, SMR10, TAP90, TG1, TG2, XL1-Blue, XS101, XS127, Y1089, Y1090hsdR, YK537, and the like, which are all described in Sambrook et al, Molecular Cloning A Laboratory Manual 2nd ed.
(4). Desulfurization Enzyme
The desulfurization enzymes of the present invention includes the following three proteins.
The first protein is a protein represented by an amino acid sequence shown in SEQ ID NO: 2, or a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, and having a function of converting DBTO2 into 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid.
The second protein is a protein represented by an amino acid sequence shown in SEQ ID NO: 4, or a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 4, and having a function of converting 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid into 2-HBP.
The third protein is a protein represented by an amino acid sequence shown in SEQ ID NO: 6, or a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 6, and having a function of converting DBT into DBTO2.
The said first, second and third proteins have a certain homology to the desulfurization enzyme DszA, DszB or DszC derived from Rhodococcus sp. IGTS8 strain, and their function as an enzyme is also identical. However, they are apparently distinct in respect of the following.
(1) DszA, DszB and DszC cannot desulfurize benzothiophene which is a desulfurization-resistant substance, but the first, second and third proteins of the present invention can do so.
(2) DszA, DszB and DszC have the desulfurization activity at around room-temperature region, but the first, second and third proteins have activity at a high-temperature region.
The desulfurization enzymes of the present invention can be prepared by using the genes encoding the said desulfurization enzymes of the present invention. Further, the desulfurization enzymes represented by amino acid sequences as shown in SEQ ID NOS: 2, 4 and 6 can also be prepared from the strains Paenibacillus sp. A11-1 (which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology under accession No. FERM BP-6025 on Jul. 22, 1997) or Paenibacillus sp. A11-2 (which was deposited with the same international depositary authority under accession No. FERM BP-6026 on Jul. 22, 1997) according to the conventional methods.
The characteristics of one example of the first protein of the present invention are as follows:
(i) Function: the first protein converts DBTO2 into 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid;
(ii) pH: as shown in FIG. 6, optimum pH: 5.5, stable pH: 5-10;
(iii) Temperature: as shown in FIG. 7, optimum temperature: 45xc2x0 C.;
(iv) Molecular weight: 120,000 (as determined by gel filtration);
(v) Inhibition of activity: the first protein is inhibited by chelating agents or SH inhibitors, but not by 2-HBP or sulfate; and
(vi) Requirement for coenzyme: NADH and FMN are required, NADPH can be substituted for NADH, but FAD cannot be substituted for FMN.
The characteristics of one example of the second protein of the present invention are as follows:
(i) Function: the second protein converts 2-(2xe2x80x2-hydroxyphenyl) benzenesulfinic acid into 2-HBP;
(ii) pH: as shown in FIG. 8, optimum pH: 8, stable pH: 5.5-9.5;
(iii) Temperature: as shown in FIG. 9, optimum temperature: 55xc2x0 C.;
(iv) Molecular weight: 31,000 (as determined by gel filtration)
(v) Inhibition of activity: the second protein is inhibited by chelating agents or SH inhibitors, but not by 2-HBP or sulfate; and
(vi) Requirement for coenzyme: no coenzyme is required.
(5) Gene Encoding Transposase
The transposase genes of the present invention encodes any of the following proteins:
(a) a protein represented by an amino acid sequence as shown in SEQ ID NO: 8,
(b) a protein represented by an amino acid sequence as shown in SEQ ID NO: 9, or
(c) a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, and having a transposase activity.
Among the transposase genes of the present invention, the ones encoding amino acid sequences set forth in SEQ ID NOS: 8 and 9 have been determined, as shown in SEQ ID NO: 7. So such genes can also be obtained by synthesizing appropriate primers on the basis of the determined sequence and carrying out PCR using, as a template, DNA prepared from Paenibacillus sp. A11-1 strain (which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology under accession No. FERM BP-6025 on Jul. 22, 1997) or A11-2 strain (which was deposited with the same international depositary authority under accession No. FERM BP-6026 on Jul. 22, 1997).
The gene encoding an amino acid sequence comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 8 or NO: 9 can be obtained by modifying the genes which encode an amino acid sequence shown in SEQ ID NO: 8 or NO: 9, according to the conventional art as of the filing date of the present application, e.g. site-directed mutagenesis (Zoller et al., Nucleic Acids Res. 10: 6487-6500, 1982)
Since this gene has transposase activity, it is possible to transfer any gene unit from a certain DNA molecule to a different DNA molecule by using this gene. By the way, it has not experimentally been determined that the polypeptide represented by an amino acid sequence as shown in SEQ ID NO: 8 or NO: 9 has transposase activity. However, there seems to be an extremely high possibility that each of the two polypeptide has transposase activity for the reasons that they have a certain homology to transposase existing in an insertion sequence IS1202, that ORFs of two polypeptides are in the reverse orientation to ORFs of desulfurization enzymes and are in a position directed to sandwich them (a structure specific for transposon), and that the direct repeat sequence (DR) and the invert repeat sequence (IR) which are specific for transposon exist at each end of SEQ ID NOS: 8 or 9.
(6) Transposase
The transposase of the present invention is selected from the group consisting of:
(a) a protein represented by the amino acid sequence as shown SEQ ID NO: 8,
(b) a protein represented by the amino acid sequence as shown SEQ ID NO: 9, and
(c) a protein comprising a deletion, substitution or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9, and having a transposase activity.
The transposase of the present invention can be prepared by using the genes encoding the above-described transposase.